1. Field of the Invention
The present invention relates to methods and compositions for tissue engineering and organ regeneration, and more particularly to methods and compositions for physiological repair of human tissues and regeneration of fully functional human organs through induction and propagation of multipotent, regenerative stem cells in vivo and in situ.
2. Description of the Related Art
The tissue engineering industry is growing at an accelerated pace owing to technological advancements in producing large scale cell culture products and biomaterials. These products are produced or synthesized ex vivo, i.e., outside an animal or human body, and then transplanted to the host for tissue repair or other therapeutic purposes.
One approach to modern tissue engineering is to implant a synthetic material into the human body as a structural scaffold for supporting the ingrowth of the tissue. For example, synthetic bone substitutes, such as α-BSM® from Etex Corp (Cambridge, Mass.), can be used for orthopedic, dental and craniofacial applications. α-BSM® is a nano-crystalline calcium phosphate that mimics the composition and structure of the mineral content of bone. When mixed with saline, it becomes a paste that can be either injected into a void or implanted as moldable putty. Once the material is in place, the hardening process is initiated by the heat of body temperature. As a result, the implant becomes a scaffold that is eventually absorbed and replaced with new bone tissue.
Another approach to tissue engineering is to utilize biological, as opposed to synthetic, matrices to provide a foundation for repair and regeneration of damaged or diseased tissues. Acellular dermal matrix is produced from fresh human cadaver skin by control process that removes the epidermis and the cells from the dermis without altering the structure of the extracellular matrix and the basement membrane complex. Wainwiright (1995) Burns 21:243–248. Acellular dermal matrix from fresh porcine skin has also been developed using a similar process in order to compensate for the lack of cadaver skin availability. Liversey et al. (1995) Transplantation 60:1–9. Recently methods have been developed by LifeCell Corp (Branchburg, N.J.) for chemically processing human skin to produce a human skin matrix. All of the skin cells are chemically removed while the bioactive, structural dermal matrix is preserved. Such a structural, biochemically intact, acellular matrix is believed to provide to a three-dimensional structural array of information that directs revascularization and repopulation in a normal regenerative response. The acellular human skin matrix serves as an allograft, i.e., a graft from a donor other than the host him/herself. The matrix is frozen in liquid nitrogen and then fractured into 100 micron particles. These small-diameter, acellular vascular grafts are being developed as an alternative to autografted blood vessels in coronary bypass procedures.
Wound healing of the skin represents a major target for tissue engineering. Repair of wound of the skin involves the timed and balanced activity of inflammatory, vascular, connective, tissue, and epithelial cells. Traditional management of large-surface or deep wounds employs the so-called dry therapy which allows the wounds to be left in a warm, dry environment to crust over. Current methods involve covering the wounds with temporary dressings and topical treatment, including antibiotics. Secondary invention, such as surgical debridement, is usually employed to remove scab or the dead tissue. For burn wounds, surgical intervention, tangential excision of a partial or full-thickness wound, is a method still widely used despite of drawbacks such as blood loss in large-surface wounds. After wound bed is prepared the wound is covered with autografts or temporary dressing to promote healing.
An autograft is harvested from the host him/herself and used as a permanent cover for the patient's own wound(s). Since the skin graft is donated by and transplanted to the patient him/herself the problems associated with immunogenicity can be avoided. The graft can be harvested from an adjacent undamaged area of the patient that matches closely in terms of texture, color, and thickness. For a small-area wound, autograft was shown to achieve good quality of healed skin by expanding the surface of the skin graft with a mesh apparatus. Tanner et al (1964) Plastic Reconstr. Surg. 34:287–292; and Richard et al. (1993) J. Burn Care Rehabil. 14:690–695. However, excessive meshing usually results in healed skin that is more susceptible to infections and which has a basket-like pattern, an undesirable result aesthetically. Alternative methods, such as the Meek island graft or sandwich graft, were also developed, which allows easier handling widely expanded autografts than meshed skin. Meek (1954) Am. J. Surg. 96:557–558; and Kreis et al. (1994) Burns 20(suppl 1) S39–S42.
However, the autograft method faces a few challenges and limitation in the treatment of patients with large surface area wounds. It has been realized that a deep burn or large-surface wounds could not be completely closed promptly after injury by using the patient's available autograft donor sites. Adequate, healthy skin donor sites are difficult to find in such patients. There is also a time limitation for harvesting the graft from the same site. Often, a delay of several weeks is necessary to wait for healing of the donor sites before harvesting them again, thus delaying healing of the “main” wound—the original wound to be treated and increasing the risk of complication. Even worse is that harvesting an autograft in fact creates a second wound in the normal healthy skin, which increases the risk of infection and fluid/electrolyte imbalance. In addition, repeated harvests of autografts from a donor wound site can result in contour defects or scarring, thereby causing disfigurement of the patient.
To find a substitute for the autologous split-thickness grafts described above a two-step procedure has been developed using composite autologous-allogenic skin replacement. Such a graft consists of a skin allograft which has its epidermis removed to serve as a dermis substitution for the patient, and autologous epidermis reconstructed in vitro with the patient's own keratinocytes. Cuono et al. (1986) Lancet 17: 1123–1124; and Compton et al. (1989) Lab. Invest. 60:600–612. The autologous epidermis is usually constructed in vitro by using the technique developed by Rheinwald and Green (1975) Cell 6:331–344. This technique consists of digesting a small biopsy of healthy skin in trypsin or in thermolysin in order to isolate keratinocytes from the basal layer of the epidermis. By culturing the autologous keratinocytes in vitro a large number of cells are available for generate enough epidermis for grafting.
This two-step approach suffers a few limitations. First, growth of cultured epidermal sheets in a laboratory needs at least 3 weeks to be achieved, thus delaying the coverage of wounds. The successful treatment demands highly sophisticated laboratories and well trained physicians/surgeons in the whole process of epidermal sheet production and grafting on the wound bed. This limitation is even more prominent in areas where such laboratory and human resources are not available, such as the battle fields and the rural areas of developing countries. Second, the reconstructed epidermal sheets need to be grafted on a clean wound bed since they are highly sensitive to bacterial infection and toxicity of residual antiseptics. Thus, proper preparation of the wound bed is critical for the survival of the fragile epidermal sheets. More significantly, although the epidermal sheet can attach to the dermis, the conjunction between these two layers is artificial relative to the natural skin. Since the regeneration of the dermal compartment underneath the epidermis is a lengthy process the skin remains fragile for at least three years and usually blisters. In addition, the aesthetic effect is usually not as good as with one obtained with a split-thickness graft.
To provide the dermal structure for the cultured epidermal sheet and promote graft takes, allogeneic skin has been used to cover the wound. After debridement, cadaver allograft is used to over the wound and the allogeneic epidermis is excised in order to maintain the allogeneic dermis on the wound. The cultured epidermal sheet is then grafted on the de-epidermized cadaver allograft. The cadaver allograft is non-vital and thus has a much-reduced antigenicity.
To overcome problems associated with delayed transplantation due to time required for culturing autologous epidermal sheet allogeneic cultured epidermal sheets were tested clinically and experimentally. Unfortunately, even though the allograft is depleted of Langerhans' cells, the rejection of the transplant by the host occurs in mice after about 2 weeks. Rouabhia (1993) Transplantation 56:259–264.
Xenogeneic grafts, i.e., tissues of other animal origin, have also been used to cover extensive wounds. Porcine skin is the most common source of xenograft because of its high similarity to human skin. Sterilization (e.g., ionizing radiation) coupled with freeze-drying seems to decrease the antigenic properties of the pigskin graft and increase its potential to inhibit bacterial growth. The xenografts are used mostly as a temporary dressing for the coverage of second-degree burns, especially after early excision. Pellet et al. (1984) in Burn Wound Coverings, Wise D L, ed., Boca Raton, CRC Press, Florida, 1:85–114.
Artificial dermal matrices have been developed to cover wounds in order to facilitate graft take of cultured epidermal sheets and to prevent rejection of xenogeneic tissues. They are used to prompt coverage of large excised full-thickness wounds, control fluid loss, and prevent infection. Examples of such artificial dermal matrices include 1) synthetic mesh composed of nylon or a polyglactic acid mesh on which fibroblasts are cultured (Rennekampff et al. (1996) J. Surg. Res. 62:288–295); 2) collagen gel made of a mixture of fibroblasts and bovine collagen (Yanna et al. (1981) Trans. Am. Soc. Artif. Intern. Organs 27:19–23); collagen sponge based on the production of a lyophilized collagen matrix in which fibroblasts are cultured and migrate (Bell et al. (1979) Proc. Natl. Acad. Sci. USA 76:1274–1278 and Bell et al. (1981) J. Invest. Dermatol. 81:S2–S10); collagen membrane (Ruszczak et al. (1998) Ellipse 14:33–44); and in vitro reconstructed skin-like products based on collagen matrix (Sabolinski (1996) Biomaterials 17:311–320).
The xenogeneic graft approach has a few limitations in clinical treatment of wounds, most prominent being immunogenicity and biocompatibility. The level of natural antibodies of the transplant host which react with organ xenotransplants increases proportionally with phylogenic distance between the xenogeneic species involved. In organ transplantation, the presence of such antibodies leads to hyperacute rejection, which occurs within minutes to hours after revascularization, and to the loss of the transplanted tissue.
To provide a large amount of keratinocytes for reconstructing autologous or allogenic epidermal sheets in vitro, great efforts have been made to cultivate human keratinocyte stem cells in culture. Keratinocytes forming the epidermal basal layer are endowed with proliferative capacity, hence they regularly undergo mitosis, differentiation and upward migration to replace terminally differentiated cornified cells that are continuously shed into the environment. The epidermis relies on the presence of keratinocyte stem cells to accomplish wound healing. The basic, essential and indispensable characteristics of a stem cell is its capacity for extensive self-maintenance with the potential for proliferative self-renewal extending for at least one lifespan of the organism. Lajtha (1979) Differentiation 14:23–34. Thus, a stem cell can divide to generate transient amplifying cells which can differentiate into one or more specialized cell types.
Keratinocyte stem and transient amplifying cells are located both in the epidermal basal layer and in the hair matrix. Lavker et al. (1983) J. Invest. Dermatol. 81:121s–127s; and Rochat et al. (1994) Cell 76:1063–1073. In preparing epidermal sheets for transplant basal keratinocytes are cultivated in culture to produce large numbers of progeny. Maintaining these stem cells in culture conditions can be challenging. The quality of the keratinocyte culture system must be carefully monitored by directly demonstrating the presence of holoclones in culture, periodical clonal analysis of a reference strain of keratinocyte both in terms of clonogenic and growth potential, and monitoring the percentage of aborted colonies. Inappropriate culture conditions can irreversibly accelerate the clonal conversion and can rapidly cause the disappearance of stem cells, rendering the cultured autograft or allograft transplantation useless.
Besides keratinocyte stem cells, other types of stem cells are cultivated in cell culture in an attempt to provide sufficient amount of cells for tissue repair or other therapeutic use. Embryonic stem (ES) cells can be cultured under proper conditions. Thomson et al. demonstrated that cells from the inner cell mass (ICM) of mammalian blastocysts can be maintained in tissue culture under conditions where they can be propagated indefinitely as pluripotent embryonic stem cells. Thomson et al. (1998) Science 282:1145–1147. Primate blastocysts were isolated from the ICM from the blastocysts and plated on a fibroblast layer wherein ICM-derived cell masses are formed. The ICM-derived cell mass were removed and dissociated into dissociated cells which were replated on embryonic feeder cells. The colonies with compact morphology containing cells with a high nucleus/cytoplasm ratio, and prominent nucleoli were selected and the cells of the selected colonies were then cultured. In this way, a primate embryonic stem cell line was established. It was observed that after undifferentiated proliferation in vitro for 4 to 5 months, these cells still maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). Thus, it is envisioned that these ES cells can be cultured and regulated under suitable conditions to coax the pluripotent cell to differentiate into cells of a particular tissue type and/or to form various organs in vitro. These cells and organs, wishfully, could be used as transplants to cure various diseases and replace dysfunctional body parts.
Although desirable, an in vitro embryonic development process is highly unpredictable. The conditions under which ES cells differentiate into a specific type of cell or organ are elusive. It has been found that to maintain cultured ES cells in their relatively undifferentiated, pluripotent state, they must both express the intrinsic transcription factor Oct4, and constitutively receive the extrinsic signal from the cytokine leukemia inhibitor (LIF). Nichols et al. (1998) Cell 95:379–391. Upon withdrawal of LIF, cultured ES cells spontaneously aggregate into a mass of cells of various tissue types. Although the programs of gene expression in these cells somewhat resemble the differentiation pathways typical of developing animals, the triggering of these programs is chaotic.
For successful organ regeneration in the clinic using stem cells cultured in vitro, a major obstacle lies in its way. Stem cells cultured in vitro must be directed to differentiate into site-specific phenotypes once they are transplanted into the lesion site. Complete deciphering of the signal needed for this process is required to guide the design of the in vitro tissue culturing conditions. Experimental data obtained by others in the art show that although multipotent human mesenchymal, mouse neural stem cells, and mouse embryonic stem cells can be grown in vitro through the addition of leukemia inhibitory factor (LIF) to the culture medium, mouse ESCs differentiate randomly in vitro and in vivo. Progress in the art has made it possible to induce differentiation of mouse ESCs into multipotent glial cell precursors in vitro and to transplant them into the brain of myelin-deficient fetal rats. However, question remains unanswered as to whether these multipotent stem cells harvested from specific tissues or differentiated from ESCs in vitro will make site-specific tissue when transplanted to injured adult tissues.
Up to date enormous amounts of money and efforts have been made in attempts to repair damaged tissue and dysfunctional organs through cultivation of stem cells in vitro. However, no successful regeneration of a fully functional human organ has been reported by using this approach. For example, treatment of wounds with in vitro cultivated keratinocyte stem cells merely closes the wound, not resulting in a full restoration of the physiological structure and function of the skin. Therefore, there exists an urgent need for innovative approaches that depart from the above strategies and provide greater benefits to human health.